4-nitrophenyl acetate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Principles of method if other than guideline:

There was a deviation from study plan. The software “Toxicita” was unable to make a calculation the value of EyC50, therefore this value was calculated by the software Excel. This deviation does not impact the integrity of study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, 4-nitrophenyl ester

IUPAC Name:

Acetic acid, 4-nitrophenyl ester

Constituent 2

Reference substance name:

4-nitrophenyl acetate

EC Number:

212-593-5

EC Name:

4-nitrophenyl acetate

Cas Number:

830-03-5

Molecular formula:

C8H7NO4

IUPAC Name:

4-nitrophenyl acetate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 4-Nitrophenyl Acetate

- Physical state: solid
- Analytical purity: 95 % w/w
- Impurities (identity and concentrations):
Phenol <5.0 % (w/w)
4-nitrophenol <5.0 % (w/w)
Acetic acid <1.0 % (w/w)
Water <5.0 % (w/w)

- Lot/batch No.: 201409
- Expiration date of the lot/batch: unlisted

- Storage condition of test material: During the study the test substance was stored in closed
container in the dark at laboratory temperature.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Preliminary test
The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (100 mg·L-1), the lowest (1 mg·L-1) test concentration and from the highest (100 mg·L-1) concentration without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory.
The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.
The analytical results showed that the test substance was not stable in test medium under the conditions of testing.

Full test
The analytical determination of the test substance concentrations was performed at the beginning of the test, after 24, 48 hours and at the end of the test in all test concentrations. The samples for analysis were taken at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory. The samples for analysis at 24, 48, 72 hours were delivered to analytical laboratory immediately after the end
of appropriate testing period. The samples were analyzed on the day of delivery.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green freshwater algae Desmodesmus subspicatus
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 20.5.2015
- Age of inoculum (at test initiation): no data
- Method of cultivation: in flasks with the test medium on indirect daylight at laboratory temperature.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed:no data

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 24 °C controlled at ± 2 °C

pH:

7.6

Nominal and measured concentrations:

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg·L-1.
The full test was performed in concentration range 1 – 88 mg·L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.39 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3.74 – 5.05 mg·L-1

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The analytical results showed that concentrations determined at the beginning of the test decreased to the end of the test more than 20%. Therefore the geometric mean concentrations should be used for evaluations and results presentation. But at the end of the test all tested concentrations were under limit of detection. The geometric mean concentrations could not be calculated for this concentrations. Therefore limit of detection was used for calculation of the geometric mean concentrations.
For the evaluation EC and NOEC values the geometric mean concentrations were used.
The value of ErC50 and confidence intervals of the ErC50 were calculated using the software “Toxicita”(Water Research Institute, Ostrava, Czech Republic).
The value of EyC50 was calculated by the software Excel. Confidence intervals of the EyC50 were calculated by the statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference substance. potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 30.3. to 2.4. 2015. are the following:
72 hour – ErC50 = 0.79 mg·L-1 (95% confidence limit: 0.70 – 0.89 mg·L-1)

Any other information on results incl. tables

Mean cell density – full test

Nominal concentration mg·L-1	Geometric mean of measured concentration mg·L-1	Mean cell density in 1 mL
		0 hours	24 hours	48 hours	72 hours
88	56.5	5000	10417	10417	4167
41	18.3	5000	12500	20833	16667
19	1.17	5000	14583	33333	108333
9	0.46	5000	20833	47917	129167
4	0.24	5000	18750	50000	135417
2	0.20	5000	25000	47917	181250
1	0.03	5000	37500	66667	310417
Control	Control	5000	54167	114583	416667

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The analytical results showed that concentrations determined at the beginning of the test decreased to the end of the test more than 20%. Therefore the geometric mean concentrations should be used for evaluations and results presentation. But at the end of the test all tested concentrations were under limit of detection. The geometric mean concentrations could not be calculated for this concentrations. Therefore limit of detection was used for calculation of the geometric mean concentrations.
For the evaluation EC and NOEC values the geometric mean concentrations were used.

72 hour – ErC50 = 4.39 mg·L-1 (geometric mean concentration)
(95% confidence limit: 3.74 – 5.05 mg·L-1)
72 hour – EyC50 = 0.10 mg·L-1 (geometric mean concentration)
(95% confidence limit: 0.06 – 0.19 mg·L-1)
72 hour – NOECr = 0.03 mg·L-1 (geometric mean concentration)
72 hour – NOECy = 0.03 mg·L-1 (geometric mean concentration)

Executive summary:

The test substance, 4-Nitrophenyl Acetate, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV.

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg·L-1. The analytical results showed, that the test substance 4-Nitrophenyl Acetate was not stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L-1 .

Based on the toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

The full test was performed in range of test substance nominal concentrations 1 – 88 mg·L-1. Samples for analytical determination were taken at the beginning, after 24 hours, 48 hours and at the end of the test.

The analytical results showed that the test substance 4-Nitrophenyl Acetate was not stable in test medium at conditions of the test. With respect to this observation the geometric mean concentrations were used for results presentation and evaluation.

Test results:

72 hour – ErC50 = 4.39 mg·L-1 (geometric mean concentration)

(95% confidence limit: 3.74 – 5.05 mg·L-1)

72 hour – EyC50 = 0.10 mg·L-1 (geometric mean concentration)

(95% confidence limit: 0.06 – 0.19 mg·L-1)

72 hour – NOECr = 0.03 mg·L-1 (geometric mean concentration)

72 hour – NOECy = 0.03 mg·L-1 (geometric mean concentration)